1. Field of Invention
This invention relates to the inhibition of the function of SHP2 by anti-SHP2 peptides, the chemical compounds 4-(2-sulfaminoethyl)benzoic acid (“SEBA”), and SEBA derivatives binding to the phosphotyrosyl phosphatase domain of SHP2.
2. Description of the Prior Art
Protein Tyr phosphorylation and dephosphorylation reactions play major role in the transduction of growth factor signals from the surface to the interior of the cell. Phosphorylation involves addition of a phosphate moiety to the hydroxyl group of the Tyr residue in proteins or peptides, while dephosphorylation refers to the removal of such phosphate from the phosphotyrosine. Phosphorylation reactions are catalyzed by Tyr kinases, while dephosphorylation reactions are catalyzed by phosphotyrosyl phosphatases (“PTPs”). Depending on their location in the cell, Tyr kinases are generally classified as cytoplasmic Tyr kinases (“CTKs”) or receptor Tyr kinases (“RTKs”). Most commonly cited CTKs include Src, focal adhesion kinase (“FAK”) and janus kinases (“JAKs”) (17, 19, 47, 72, 73), while the most commonly studied RTKs are the epidermal growth factor receptor (“EGFR”) family, the platelet growth factor receptor (“PDGFR”) family, the fibroblast growth factor receptor (“FGFR”) family and the insulin receptor (30, 45, 82, 96). Similarly, PTPs are classified as cytoplasmic or receptor type depending on their location in the cell (7, 8, 66, 93). This invention focuses on SHP2, the cytoplasmic PTP that has been shown to positively modulate RTK signaling, particularly, signaling by the epidermal growth factor receptor (“EGFR”) family.
SHP2 possesses two tandemly-arranged Src homology 2 (“SH2”) domains in the N-terminal region and a PTP domain in the C terminal region (32, 33). Both of these domains are essential for its biological activity (14, 32, 33, 78, 79, 86). It also possesses two tyrosine phosphorylation sites and a proline-rich motif (—PXXP—) in the extreme C-terminal region (10, 29, 69). The SHP2 protein assumes a “closed conformation” when inactive and an “open conformation” when active. In the closed conformation, the N-SH2 domain interacts with the PTP domain thus physically impeding the PTP domain from binding to target substrates. Upon engagement of the N-SH2 domain with phosphotyrosyl residues, the protein assumes an open conformation, relieving the PTP domain and rendering the enzyme active (41). In the N-SH2 domain, Asp61 and Glu76 are the mediators of this interaction. As a result, mutation of these residues creates a constitutively active SHP2 (70). Recent findings show that constitutively active SHP2 mutatants naturally occur in Noonan Syndrome-associated leukemia (87-89).
In the catalytic process of most PTPs, including SHP2, Asp in the WPD loop and the conserved residues in the signature motif play critical roles in the dephosphorylation reaction (34). In SHP2, Asp425 acts as a proton donor for the leaving phenolate group of the substrate and as an acceptor during the hydrolysis of the cysteinyl phosphate intermediate. The Cys residue conducts a nucleophilic attack on the phosphate moiety, while the Arg residue (positively charged) serves as a coordinator of the negatively charged phosphate group on the substrate. Basically, the Arg residue mediates substrate binding to the active site of the enzyme, whereas the Cys and Asp residues catalyze the dephosphorylation reaction. In addition, Thr466 is essential for SHP2 catalysis (61), but its specific role has not been established. Mutation of this residue to Ala provides a new substrate-trapping mutant of SHP2 (61). SHP2 is a unique PTP that positively modulates RTK signaling in vertebrates (32, 33, 35, 38). Interaction of SHP2 with Tyr-phosphorylated RTKs and adaptor proteins such as Gab1 and 2 and FRS1 and 2 through its SH2 domains is important for its function (28, 38, 51, 55, 80, 92). This interaction recruits SHP2 to its substrate microdomain. Deletion of the N-SH2 domain or mutation of critical residues in the active site of the enzyme such as Cys459 to Ser or Arg465 to Glu inactivates SHP2, suggesting that both domains are important for its function. These mutants have served as dominant-negative counterparts (“DN-SHP2”) in SHP2 studies as their expression inhibits the activation of the Ras-ERK (ERK for extracellular signal regulated kinase 1 and 2) and the PI3K-Akt (PI3K for phosphatidylinositol 3-kinase) signaling pathways downstream of RTKs (5, 6, 53, 54, 92).
SHP2 enhances the signaling potential of the EGFR family of RTKs. The EGFR family comprises four members (“EGFR1-4”) and represents one of the most extensively studied RTKs in the mammalian system (30, 48, 57, 58, 82, 95). The human homologues are called HER1, HER2, HER3 and HER4. Because HER1 is commonly known as EGFR, this abbreviation will be used hereinafter. All family members are composed of an extracellular ligand-binding region, a single-pass transmembrane region and a cytoplasmic region containing a tyrosine kinase domain (except HER3 that has dysfunctional kinase domain) and tyrosine autophosphorylation sites. A family of a dozen growth factors, including epidermal growth factor (“EGF”), transforming growth factor-α (“TGFα”) and neuregulins, activate these receptors by binding to the extracellular region (except HER2 that does not require ligand binding for activation). The binding of a cognate ligand induces homo- or hetero-dimerization of EGFR molecules leading to the activation of their tyrosine kinase domain and autophosphorylation of specific tyrosine residues in the C-terminal region or Tyr phosphorylation of downstream substrates (24, 26, 28, 43, 51, 59, 71). Phosphorylated tyrosine residues serve as binding sites for SH2 domain-containing signaling molecules such as the Grb2-SOS complex, the phosphatidylinositol 3-kinase (“PI3K”), SHP2 and others (74), which leads to formation of multimeric signaling complexes. At least two known functions are effected by these interactions: recruitment of enzymes to substrate micro domains (e.g. Grb2-SOS) and induction of enzyme activity (egs. PI3K, SHP2) (2, 12, 33, 38, 55, 59, 80, 83). The immediate response to stimulation with EGF or the related ligands is the activation of the Ras-ERK (ERK for extracellular regulated kinase 1 and 2) and the PI3K-Akt (Akt is sometimes referred to as protein kinase B) signaling pathways, which induce mitogenesis and cell survival, respectively. Structural and biochemical analyses have shown that HER2 does not require ligand binding for activation of its tyrosine kinase domain (36). Thus, HER2 can potentially homodimerize in the absence of ligand (especially under conditions of overexpression) or heterodimerize with ligand-stimulated family members. Generally, HER2 is regarded as the preferred partner of heterodimerization with the other family members most probably due to the unconstrained conformation of the dimerization arm of the extracellular region (36). In case of HER3, heterodimerization with other family member is the only mechanism for its activation because it lacks a functional Tyr kinase domain (42).
Regulation of cell shape and morphology by SHP2 has been correlated with its effect on actin cytoskeletal reorganization. Cells expressing dominant-negative SHP2 manifest an increased level of actin stress fiber formation and assume a flattened morphology, whereas cells expressing wild-type SHP2 show a low level of actin stress fiber formation and a more polarized morphology (46, 50, 84). These effects of SHP2 have been ascribed to its negative regulatory role of the Rho GTPase (25, 52, 84).
SHP2 suppresses cell adhesion and enhances motility (46, 50, 56, 102). However, the molecular basis underlying these effects is not clear. Cell migration requires a coordinated cycling between adhesion and detachment. In a tissue or in confluent monolayer of cultured cells, movement of a single cell in a defined direction involves modulation of its interaction with neighboring cells and the extracellular matrix (“ECM”). Adhesion of cells to the ECM is mediated by cell surface receptors called integrins. Integrin-ECM interaction recruits cytoplasmic tyrosine kinases (“CTK”) such as FAK, related focal tyrosine kinase (“RFTK”) or Pyk2 and Src (72, 81) to the plasma membrane. Autophosphorylation of FAK initiates the cascade of tyrosine phosphorylation reactions by recruiting Src to focal adhesions (72, 81). The adaptor proteins p130Cas and paxillin are the other major proteins known to participate in focal adhesion formations. The SH3 and Src-binding domains of p130Cas mediate interaction with FAK and Src, which in turn mediate Tyr phosphorylation (65, 76). Paxillin possesses lucine-rich motifs that promote its interaction with FAK and the other focal adhesion protein vinculin (21, 27). Paxillin also possesses Tyr phosphorylation sites that mediate SH2 domain interactions (99). The net effect is the aggregation of integrins, the formation of multi-protein complexes and the maturation of focal complexes to focal adhesions (56, 100). SHP2 has been repeatedly implicated in down regulating focal adhesions, but the molecular mechanism is not clear.
SHP2 mediates cell transformation induced by v-Src (39) and the constitutively active form of fibroblast growth factor receptor 3 (K650E-FGFR3) (6). In K650E-FGFR3-induced transformation, SHP2-mediated activation of the Ras-ERK and PI3K-Akt pathways was essential. Recently, it was demonstrated that SHP2 promotes K650E-FGFR3-induced transformation not only by promoting the activation of the Ras-ERK and PI3K-Akt pathways, but also by modulating the interaction of the actin cytoskeleton with adherens junction (23). The recent discovery of gain-of-function SHP2 mutations in Noonan syndrome and associated leukemia (15, 62, 89) and the subsequent demonstration of the development of lymphoid hyperplasia in transgenic mice expressing gain-of-function SHP2 mutants (9, 62) further support the importance of SHP2 in cancer.
The driving force behind this invention is that SHP2 is an essential transducer of cell transformation induced by Tyr kinase oncogenes, which in turn suggests that it promotes tumor growth. Recent reports have uncovered molecular mechanisms by which the phosphatase activity of SHP2 promotes signaling by Tyr kinases, particulary by the EGFR. The development of a substrate-trapping mutants of SHP2 was a breakthrough for understanding its molecular mechanism (4). Substrate trapping refers to the production of mutant phosphatases that retain substrate-binding ability or acquire enhanced substrate-binding ability, but are devoid of enzyme activity (34). Thus, by introducing mutations in the active site of SHP2, an efficient substrate trapping mutant termed DM-SHP2 (DM for double mutant) was developed (4). Using the DM-SHP2 as a reagent, the first biological substrate of SHP2, the EGFR, was identified and characterized. Subsequently, a molecular mechanism for SHP2 in promoting EGFR signaling was described. It was demonstrated that SHP2 promotes Ras activation by interfering with the process of Ras inactivation catalyzed by the Ras GTPase activating protein (RasGAP). Inhibition is achieved through the dephosphorylation of Tyr992 of the EGFR, which serves as RasGAP binding site (5).
Substrate-trapping and mass spectroscopic analysis showed that α-catenin also is a biological substrate of SHP2 (23). Tyr phosphorylation of α-catenin enhances its translocation to the plasma membrane and its interaction with β-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Dephosphorylation of α-catenin by SHP2 suppresses intercellular adhesion and increases the cytosolic pool of β-catenin and its subsequent translocation to the nucleus (23) where it acts as a transcription factor for mitogenic genes such as cyclin D1 and c-myc (1, 13). In addition, inhibition of α-catenin enhances activation of the Ras-ERK and the PI3K-Akt pathways (23, 97), induces hyperproliferation of skin epithelium (97) and promotes cell transformation (22, 63). Therefore, SHP2 mediates β-catenin activation downstream of RTKs bypassing the need for Wnt ligand stimulation (23). The previously held notion was that β-catenin is activated downstream of the frizzled (FZ) and low-density lipoprotein related protein (“LRP5/6”) coreceptors following Wnt ligand stimulation (18, 49, 60).
In EGFR and HER2, SHP2 dephosphorylates negative-regulatory phosphorylation sites that serve as RasGAP binding sites. By doing so, SHP2 promotes the Ras-ERK and the PI3K-Akt signaling pathways (5). In α-catenin, SHP2 dephosphorylates a phosphotyrosyl that mediates interaction with β-catenin (23). This leads to suppression of adherens junction-mediated cell-cell interaction, an increase in cytosolic β-catenin pool and subsequent translocation of β-catenin to the nucleus where it acts as a transcription factor for mitogenic genes. Thus, through SHP2, EGFR and HER2 can activate, not only the Ras-ERK and the PI3K-Akt pathways, but also β-catenin signaling. Therefore, mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. These findings suggest that SHP2 could be a potential target for cancer treatment.
Examples that highlight the importance of SHP2 in growth factor signaling, cell transformation and cancer development are provided below. These results were based on inhibition of SHP2 either by dominant-negative SHP2 (DN-SHP2) expression or small interfering RNA (Si-RNA)-mediated ablation of the SHP2 protein. As known in the art, DN-SHP2 expression refers to ectopic expression of a dysfunctional mutant protein that competes with the endogenous counterpart and inhibits function. The controls for these experiments were vector alone and the wild type form of SHP2 (WT-SHP2). The indicated breast cancer cell lines were infected with retrovirus expressing vector alone, WT-SHP2 or DN-SHP2, and stable lines from each group were seeded in soft agar, a commonly used assay for testing anchorage-independent growth (3, 23). The MCF10A, the immortalized normal breast cell line, was used as a negative control for anchorage-independent growth. Expression of DN-SHP2 inhibited colony formation by all of the breast cancer cells used in this experiment, while expression of vector alone or WT-SHP2 did not cause any change (FIG. 1), As expected, the negative control MCF10A cells could not form colonies in soft agar during anchorage-independent growth studies. In addition, expression of DN-SHP2 modestly suppressed cell growth in all of the cells used in this study (FIG. 2). Together, these results show that SHP2 is important for cell proliferation and anchorage-independent growth of breast cancer cells.
The importance of SHP2 in cell transformation and growth factor signaling was further investigated by Si-RNA-mediated ablation of the SHP2 protein in the BT474 breast cancer cells that overexpress the HER2 oncogene. As known in the art, expression of Si-RNA in cells inhibits the translation of the corresponding messenger RNA (mRNA) by hybridizing to a specific complementary region within the mRNA. For the current work, stable cell lines harboring the SHP2 Si-RNA were produced by infection of the BT474 cells with retroviruses expressing the SHP2 Si-RNA under the control of a tetracycline-inducible system (BD Biosceinces). These cells express the SHP2 Si-RNA only when they were treated with tetracycline. Parent BT474 cells were used as controls (control) in these experiments. In addition, cells harboring the SHP2 Si-RNA (non-Si-RNA), but not treated with tetracycline were used as controls for the effect of retroviral infection and gene integration. Therefore, the experimental groups used in these studies included controls, non-Si-RNA and Si-RNA cells. Si-RNA-mediated ablation of the SHP2 protein induced reversion of the BT474 cells to a normal phenotype that compares with the cobblestone-like appearance of the MCF10A, the immortalized normal breast epithelial cell line (FIG. 3). It also inhibited anchorage independent growth in soft agar (FIG. 4) and led to re-differentiation of the BT474 cells back to normal as evidenced by acini formation in laminin-rich basement membrane (LRBM) cultures (FIG. 5). It should be noted that Si-RNA cells die in soft agar, but re-differential to normal under adherent two dimensional and LRBM cultures. These results demonstrate that SHP2 is important for the maintenance of the transformation phenotype of breast cancer cells. In addition, they show that interference with SHP2 function leads to re-differentiation of breast cancer cells to a normal phenotype.
In addition to morphological and growth behavior changes, Si-RNA-mediated ablation of the SHP2 protein inhibited EGF-induced signaling. The indicated cells were grown to subconfluency, serum starved for about 12 hours and then stimulated with 10 ng/ml EGF for varying time points. Lysates prepared from these cells were separated by denaturing polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane and analyzed by western blotting with antibodies that recognize the activated forms of ERK1/2 and Akt. Si-RNA-mediated ablation of the SHP2 protein inhibited EGF-induced activation of these proteins (FIG. 6), suggesting that SHP2 is required for mitogenic and cell survival signals in breast cancer cells. Therefore, SHP2 has the potential to serve as a new drug target for the treatment of breast cancer.